Polymer beads

ABSTRACT

Azlactone-functional polymer beads are useful reactive supports for the attachment of functional materials to provide novel adduct beads. The adduct beads are useful as complexing agents, catalysts, reagents, and as enzyme or other protein-bearing supports. Novel carboxylate-functional polymer beads, are intermediates in the preparation of the azlactone-functional beads. 
     Azlactone-functional beads have units of the formula: ##STR1## wherein R 1  is H or CH 3 , 
     R 2  and R 3  independently can be an alkyl group having 1 to 14 carbon atoms, a cycloalkyl group having 3 to 14 carbon atoms, an aryl group having 5 to 12 ring atoms, an arenyl group having 6 to 26 carbon and 0 to 3 S, N, and nonperoxidic O heteroatoms, or R 1  and R 3  taken together with the carbon to which they are joined can form a carbocyclic ring containing 4 to 12 ring atoms,and 
     n is an integer 0 or 1.

FIELD OF THE INVENTION

This invention relates to azlactone-functional polymer beads. In anotheraspect, carboxylate-functional polymer beads, which are intermediates inthe preparation of the azlactone-functional beads, are provided. Theazlactone-functional polymer beads are useful reactive supports for theattachment of functional materials to provide novel adduct beads. Theadduct beads are useful as complexing agents, catalysts, reagents, andas enzyme or other protein-bearing supports. In additional aspects,methods of preparation of the three types of beads are disclosed.

BACKGROUND OF THE INVENTION

The attachment of useful materials such as catalysts, reagents,chelating or complexing agents, and proteins to insoluble supports iswell-known. With the attending advantages of ease of removal andrecovery from the system, e.g., by simple filtration, regeneration (ifnecessary), and recycling coupled with the increased utilization ofcontinuous flow systems in both general chemical processing anddiagnostic monitoring procedures, supported materials are ubiquitous intoday's technology. One indication of this is the listing of"Polymer-Supported Reagents" as a separate heading in the GeneralSubjects Index of Chemical Abstracts beginning in 1982.

Concerning the nature of the insoluble support material, both inorganicpolymers (notably silica gel and alumina) and organic polymers have beenutilized. Factors, however, such as increased capacity because of betterporosity (especially with the so-called "gel-type" polymers which swellsomewhat and allow relatively free access by solvent and solute to thebound functionality within the support) and better control of the polarnature of the support (by selection of appropriate comonomers), whichhas been shown to directly affect reaction rate, have led to a generalpreference for the organic polymer supports. Polystyrene has been thesolid support material most extensively utilized.

The attaching functionality for polystyrene supports most often utilizedhas been the chloromethylphenyl group. These reactive, solid supportsare the so-called "Merrifield resins", so named for R. B. Merrifield (J.Am. Chem. Soc., 85, 2149 (1963)) who received the Nobel Prize inChemistry in 1984 for these and other achievements. Merrifield resinsare extremely useful for conducting solid phase peptide syntheses, buttheir broad utilization as reactive, solid supports is limited becauseof the relative nonpolarity of the hydrophobic polystyrene backbone, anoftentimes unpredictable attaching reaction which involves nucleophilicdisplacement of chloride ion, and a relatively low capacity of reactablechloromethylphenyl groups per gram of polymer. The chloromethylphenyland other reactive functionalities are discussed by N. K. Mathur, C. K.Narang, and R. E. Williams, "Polymers as Aids in Organic Chemistry",Chapter 2, Academic Press: New York (1980).

The present state of reactive, insoluble supports may be summarized bythe statement that no one support is broadly suitable for the manyapplications of solid-supported materials. The spectrum of propertiesrequired varies tremendously depending on the end-use, which includessuch diverse applications as mediating organic synthetictransformations, removing precious metals from sea water or heavy metalcontaminants from industrial effluants, utilizing supported metals ascatalysts for conducting organic reactions and polymerizations,resolving optical isomers, and attaching biomacromolecules.

Azlactones have not been previously utilized as attaching groups oninsoluble supports. Azlactones have, however, been proposed to be usefulin two instances.

U.S. Pat. No. 4,070,348 teaches the preparation of water-swellable,crosslinked bead copolymers. The copolymers are reactive with proteinsprimarily by the inclusion of oxirane groups which are the only reactivegroups claimed. Several "activated carboxyl groups" (col. 4; line 42),however, are listed including a 2-alkenyl azlactone,2-isopropenyl-4,4-dimethyl-oxazolone-5 (col. 5; lines 2-3), and reactionof this compound with a primary amino group of a protein is depictedschematically (col. 5; lines 6-14). No additional information orenabling disclosure is given about incorporation of the azlactone into ahydrophilic, crosslinked bead copolymer or reaction of anazlactone-functional insoluble support with a protein or any otherfunctional material. The crosslinked, bead copolymers of U.S. Pat. No.4,070,348 are all prepared purposely in essentially an anhydrouscondition, i.e. with care being taken to exclude water.

L. D. Taylor, et al., Makromol. Chem., Rapid Commun., 3, 779 (1982) haveproposed azlactones to be useful as reactive groups on polymericsupports. Only the bulk homopolymerization of2-vinyl-4,4-dimethylazlactone to form a polymeric "plug" is described.No mention of crosslinking and generation of polymeric beads is given.Furthermore, described at some length is the susceptibility of thepoly(azlactone) to hydrolysis, i.e., ring-opening reaction with water[equation (1)]. Hydrolysis is regarded as being very facile, occurringeven with traces of moisture often present in organic solvents for thehomopolymer, as follows: ##STR2## Based on this account of thepropensity toward hydrolysis, it is entirely unexpected that anazlactone-functional support could be selectively reacted with afunctional material in aqueous media.

SUMMARY OF THE INVENTION

Briefly, the present invention provides crosslinked, hydrophilicazlactone-functional polymer beads.

In another aspect, the present invention provides crosslinked,hydrophilic carboxylate-functional polymer beads which are intermediatesin the preparation of the azlactone-functional polymer beads of theinvention. The hydrophilic carboxylate-functional polymer beads haveadditional utility, for example, as ion exchange resins and ashydrophilic adsorbents.

In a further aspect, the present invention provides novel adduct beadswhich are produced by a ring opening reaction between theazlactone-functional polymer beads of the invention and functionalmaterials. The adduct beads are useful as complexing agents, catalysts,reagents, and as enzyme- and other protein-bearing supports.

The present invention provides a novel method for the preparation of thethree types of beads of the invention. The carboxylate-functionalpolymer beads are prepared as the polymerization reaction product of:

(i) optionally, at least one free radically addition polymerizable,water soluble monomer,

(ii) at least one water-soluble salt of an N-(meth)acryloylamino acid,and

(iii) at least one crosslinking monomer.

Reaction of a cyclization agent and the carboxylate-functional polymerbeads of the invention provides the azlactone-functional polymer beads.

Reaction of the azlactone-functional polymer beads of the invention withfunctional materials capable of reacting with the azlactone ring (i.e.,by a ring-opening reaction) provides the adduct beads of the invention.We have discovered that this adduct-forming reaction occurs to a highdegree with a dissolved nucleophile in water solution, especially whenthe nucleophile is primary amine-functional. This selectivity ofreaction is even more surprising when one considers that theconcentration of the amine nucleophile is most often substantially lowerthan that of the water solvent. Before the present invention, it wasthought that azlactone groups would predominantly react with water,i.e., hydrolyze, rather than with a dissolved nucleophile.

The hydrophilic or hydrophobic nature of an organic polymer support isextremely important in determining its utility. An obvious advantage ofa hydrophilic support is that many of the operations of supportedmaterials are conducted in aqueous media. Water is virtually theexclusive solvent for conducting precious or noxious metal ion removal,diagnostic monitoring of components of biofluids and biosystems, as wellas a number of chemical reactions, and it is oftentimes advantageous toutilize a polymer support which will swell in water. The water solventcan facilitate the additional encounter and interaction of a solute andreactive groups within the hydrophilic support as well as at thesupport-water interface.

Hydrophilic polymer-supported materials find use and are beneficial innon-aqueous systems as well. Functional groups which imparthydrophilicity are highly polar in nature, and supported materialfunctions which are sensitive to solvent effects will be tremendouslyaffected, especially in terms of rate, by the polarity of the polymerbackbone. The importance of the polymer backbone in determining thelocal environment for a supported material has been noted by H.Morawetz, J. Macromol. Sci.--Chem., A-13, 311 (1979) and is hereinincorporated by reference.

In this application:

"acryloyl" means not only 1-oxo-2-propenyl but also1-oxo-2-methyl-2-propenyl resulting from methacryloylation reactions;

"alkyl" means the monovalent residue remaining after removal of ahydrogen atom from a saturated linear or branched chain hydrocarbonhaving 1 to 14 carbon atoms;

"aryl" means the monovalent residue remaining after removal of onehydrogen atom from an aromatic or heteroaromatic compound which canconsist of one ring or two fused or catenated rings having 5 to 12 ringatoms which can include up to 3 heteroatoms selected from S, N, andnonperoxidic O. The carbon atoms can be substituted by up to threehalogen atoms, C₁ -C₄ alkyl, C₁ -C₄ alkoxy, N,N-di(C₁ -C₄ alkyl)amino,nitro, cyano, and C₁ -C₄ alkyl carboxylic ester;

"arenyl" means the monovalent residue remaining after removal of ahydrogen atom from the alkyl portion of a hydrocarbon containing bothalkyl and aryl groups having 6 to 26 carbon and heteroatoms (wherein theheteroatoms are up to 3 S, N, and nonperoxidic O atoms);

"azlactone" means 2-oxazolin-5-one groups of Formula I and2-oxazin-6-one groups of Formula II; ##STR3##

"parts" means parts by weight unless otherwise specified; and

"carboxylate" means ##STR4## wherein M is hydrogen, ammonium, or analkali metal such as Li, Na, or K.

Structures and formulae depicted between parentheses are partialstructures of crosslinked polymers.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides azlactone-functional beads having unitsof the formula: ##STR5## wherein R¹ is H or CH₃,

R² and R³ independently can be an alkyl group having 1 to 14 carbonatoms, a cycloalkyl group having 3 to 14 carbon atoms, an aryl grouphaving 5 to 12 ring atoms, an arenyl group having 6 to 26 carbon and 0to 3 S, N, and nonperoxidic O heteroatoms, or R² and R³ taken togetherwith the carbon to which they are joined can form a carbocyclic ringcontaining 4 to 12 ring atoms, and

n is an integer 0 or 1.

This invention also provides a novel intermediate in the preparation ofthe azlactone-functional beads of the invention. The novel intermediateis a carboxylate-functional bead having the formula ##STR6## wherein R¹,R², R³, and n are as previously defined, and

M is a water-solubilizing cation such as hydrogen, ammonium, or analkali metal such as lithium, sodium, and potassium.

Also provided by this invention are adduct beads having the formula##STR7## wherein

R¹, R², R³, and n are pr defined, X can be --O--, --S--, --NH, or##STR8## wherein R⁴ can be alkyl or aryl, and G is the residue of HXGwhich performs the complexing, catalyzing, or reagent function of theadduct beads.

HXG can be a protein (e.g. enzyme), dye, catalyst, reagent, and thelike.

The polymer beads of the invention are provided according to the processdepicted in FLOW CHART I, below. ##STR9##

The crosslinked hydrophilic, azlactone-functional polymer beads ofFormula V are prepared by a novel two-step process. In the first stepthe following group of monomers is subjected to a free radicalpolymerization reaction:

(i) 0 to 98 parts of at least one water soluble monomer;

(ii) 1 to 99 parts of at least one water soluble salt ofN-(meth)acryloylamino acid; and

(iii) 1 to 30 parts of at least one crosslinking monomer.

The product of the above polymerization reaction is the crosslinked,hydrophilic, carboxylate-functional beads of Formula IV. The second stepof the process involves treating the carboxylate-functional beads with acyclization agent to form the azlactone-functional beads of theinvention.

The degree of hydrophilicity of the polymer support is largelydetermined by the amount of water soluble monomer employed, althoughsome limited hydrophilicity is imparted by the functional groupscreated, i.e., amide-amide, amide-ester, or amide-thiolester with amine,alcohol, or thiol nucleophiles (HXG as defined above), by thering-opening, azlactone/nucleophile reaction (step 3). Therefore, in thestrictest sense of the present invention, inclusion of a water solublemonomer is optional. Suitable water soluble monomers exhibit asolubility of at least 3 parts in 100 parts water. Preferred monomersinclude vinyl group-containing and acryloyl group-containing compounds.A representative list of such monomers includes acrylamide,methacrylamide, N,N-dimethylacrylamide, diacetoneacrylamide,N-vinylpyrrolidone, hydroxyethyl methacrylate,2-acrylamido-2-methylpropanesulfonic acid and its salts,N-(3-methacrylamidopropyl)-N,N,N-trimethylammonium salts,N,N-dimethylaminoethyl methacrylate, acrylic acid, methacrylic acid,itaconic acid, and combinations thereof. Preferred water solublemonomers are N,N-dimethylacrylamide and N-vinylpyrrolidone.

The N-acryloylamino acid salt monomers include ammonium, sodium,potassium, and lithium salts of N-acryloylamino acids of Formula VII andare prepared by mixing (at <30° C.) equal molar quantities of aqueoussolutions of, for example, ammonium hydroxide, sodium hydroxide,potassium hydroxide, or lithium hydroxide and the Formula VII compounds.##STR10## wherein R¹, R², R³, and n are as previously defined.

The N-acryloylamino acid compounds are well-known and can be readilysynthesized. For Formula VII compounds in which n=0, either the sodiumsalt of the appropriate amino acid can be acryloylated, for example,according to K. Huebner, et al., Makromol. Chem., 11, 109 (1970) or,more efficiently, by the method of assignee's co-pending patentapplication U.S. Ser. No. 865,190 (filed Dec. 23, 1985) which involvesthe one-pot transformation of a ketone into an N-acryloylamino acid;both procedures are herein incorporated by reference. For Formula VIIcompounds wherein n=1, a useful preparation is the transformation of3,3-disubstituted acrylic acids as disclosed by D. I. Hoke, et al., J.Polym. Sci.: Polym. Chem. Ed., 10, 3311 (1972) which is alsoincorporated by reference.

Insolubilization is a necessary condition for easy removal of thesupport beads from the system. This is accomplished by inclusion of amonomer which contains a plurality of polymerizable groups and whoseparticipation in a polymerization reaction results in the physicaljoining of polymer backbones or crosslinking. Crosslinking is alsodesirable in polymer-supported materials because the mechanicalstability is generally substantially enhanced and some degree of controlof bead size can be exercized by manipulation of the level ofcrosslinking, i.e., in general for a given polymerization condition, thegreater the amount of crosslinker the smaller the bead size. The degreeof crosslinking depends primarily on the intended use of the supportmaterial. In all instances the polymers are insoluble in all solventsand possess a molecular weight which is essentially infinite. Forapplications requiring fairly high capacities and involving relativelysmall solute reaction partners which can diffuse into the swollenpolymer support, low to moderate degrees of crosslinking are desired.According to D.C. Sherrington, Br. Polym. J., 16, 164 (1984),crosslinked swellable supports (referred to as "gel-type" polymers)result from inclusion of from 1 to 20 parts of a difunctional monomer.For applications which allow low capacities, low degrees of physicalexpansion due to swelling (as in certain operations conducted inconfined flow systems such as columns), and which involve large solutes,e.g., biomacromolecules, which cannot because of their large sizediffuse into the polymer network, highly crosslinked systems resultingfrom copolymerization of more than 20 parts of a difunctional monomerare utilized. These are so-called "macroporous" polymers, andsolute/support reactions occur primarily at the solvent/supportinterface.

Suitable crosslinking monomers include α,β-unsaturated esters such asethylene diacrylate and ethylene dimethacrylate, and α,β-unsaturatedamides, such as methylenebis(acrylamide), methylenebis(methacrylamide),N,N'-diacryloyl-1,2-diaminoethane,N,N'-dimethacryloyl-1,2-diaminoethane, and reaction products of2-alkenyl azlactones and short chain diamines such as those representedby Formulae VIII and IX: ##STR11## The crosslinking monomers should beat least sparingly soluble in water but need not be as water soluble asdefined for the water soluble monomer component. This is not generally aproblem for the preparation of gel-type polymers because relativelysmall proportions of the crosslinking monomers are utilized withrelatively large quantities of water solvent, and often the watersoluble monomer component, especially N,N-dimethylacrylamide andN-vinylpyrrolidone, will facilitate solution of the crosslinkingmonomer. For macroporous polymers, however, in which the concentrationis >20 parts it may be necessary to add a co-solvent which willfacilitate dissolution of the crosslinking monomer. Suitable co-solventsinclude N,N-dimethylformamide, N,N-dimethylacetamide,N-methylpyrrolidone, and dimethylsulfoxide.

The technique of polymerization employed in the present invention isoften referred to as "reverse-phase" or "inverse" suspensionpolymerization, and a general discussion of this technique by M. Munzer,et al., "Suspension Polymerizations from Non-Aqueous Media", in"Polymerization Processes" edited by C. E. Schildknecht and I. Skeist,Wiley-Interscience: New York, pp. 123-124 (1977) is herein incorporatedby reference. The reversal of the normal suspension polymerizationtechnique (in which water is the usual suspending medium) is necessarybecause the monomers of the present invention are soluble in water andtherefore require a water immiscible suspending medium.

The primary purpose of the suspending medium, besides functioning as aninert medium for dispersion of the polymerizable phase, is to dissipatethe heat generated in the polymerization reaction. An importantcharacteristic of the suspending medium is its density. In order toobtain spherical polymer beads of uniform size, the beads, once formed,should not exhibit a tendency to sink or float in the suspending medium.Therefore, the suspending medium and aqueous phases should be ofapproximately the same density.

The actual polymerization occurs in individual droplets of watercontaining the dissolved monomers and initiator. The droplets are formedand maintained in the suspending medium by vigorous agitation, and theirsize is controlled by the addition of various suspending agents whichare surface active molecules that generally contain both hydrophobic andhydrophilic parts.

In and of itself, the polymerization step is not a novel aspect of thepresent invention. As is apparent to one skilled in the art, the natureof the suspending medium, the amount of water employed, the initiationsystem, the amount of crosslinking agent, the stirring rate, and thesuspending agent are all essentially independent and important variablesthat determine the shape and size of the polymeric beads. While notwishing to be bound by any particular set of polymerization conditions,we have found the reverse-phase suspension polymerization proceduredescribed by G. L. Stahl, et al., J. Org. Chem, 44, 3424 (1979) to beexceedingly useful. In that procedure a mixture of heptane and carbontetrachloride is utilized as the suspending medium; the initiationsystem is the ammoniumpersulfate/N,N,N',N'-tetramethyl-1,2-diaminoethane redox couple; thestirring rate is 300 rpm; and the suspending agent is sorbitansesquioleate. Substitution of the various components by comparablematerials can certainly be made, and such substitutions would not beoutside the spirit and scope of the present invention.

Step two of the process of the invention consists of conversion of thecarboxylate-functional beads into azlactone-functional beads. This isaccomplished using a cyclization agent. A cyclization agent is a reagentthat can react with the carboxylate-functional beads to form anintermediate adduct which is susceptible to intramolecular attack by theamide carbonyl group to form azlactone groups according to Flow ChartII. This susceptibility is chiefly accomplished by forming a goodleaving group (.sup.⊖ O(CA) below) for the nucleophilic attack by thecarbonyl. ##STR12## wherein R¹, R², R³, and n are as defined above.

(Structures and formulae depicted between parentheses are partialstructures of crosslinked polymers depicting side chains that activelyparticipate in the cyclization reaction. Use of brackets has the usualmeaning of chemical intermediates or activated complexes. Dotted linesmean partial bonds, and α means partial ionic charges.)

Useful cyclization agents for transformation of thecarboxylate-functional beads include, by way of example, aceticanhydride, trifluoroacetic anhydride, and alkyl chloroformates such asmethyl, ethyl, and isopropyl chloroformates. Carbodiimides such asN,N'-dicyclohexylcarbodiimide can be effectively utilized but require anadditional step of acidifying the carboxylate-functional beads to formcarboxyl-functional beads which can then be cyclized toazlactone-functional beads using the carbodiimide reagent. To facilitateunderstanding of the cyclization step of the invention, theintermediates that would result by employing the aforementionedcyclization agents are depicted below in order of mention. ##STR13##

The progress of the cyclization reaction can be easily monitored byexamination of the infrared spectrum of the polymer beads. Appearance ofa carbonyl stretching absorption at about 1820 cm⁻¹ is evidence ofazlactone groups. Indeed, one reason azlactone groups are so useful aslinkages for covalent attachment to polymers is the ability to monitorreactions by observation of this infrared absorption, either theappearance of it in the synthesis of the azlactone-functional beads orthe disappearance of it in the subsequent reaction with a functionalmaterial. This absorption is strong, very characteristic of azlactones,and located in a region of the infrared spectrum where essentially noother common absorptions are observed. This is a decided advantage overother linking functional groups such as the chloromethylphenyl andoxirane which lack these unique features in their infrared spectra. Aconvenient analytical method for monitoring attaching reactions reallydoes not exist with these latter groups.

Because of its low cost and availability, acetic anhydride is apreferred cyclization agent. Typically, the carboxylate-functional beadsare covered with acetic anhydride, and the mixture is warmed attemperatures from 40°-100° C., preferably 80°-100° C., for a period of2-24 hours. After the cyclization reaction, the polymer beads arefiltered. What also makes acetic anhydride particularly preferred isthat the by-product of cyclization, the alkali metal acetate salt, isfairly soluble in acetic anhydride and can easily be removed from theazlactone-functional beads. The beads can then be dried directly or, asis often conducted, subjected to a series of washing operations withnon-reactive organic solvents such as acetone, toluene, ethyl acetate,heptane, and chloroform prior to drying.

The crosslinked, hydrophilic, azlactone-functional polymer beads of theinvention have now been formed and are ready for reaction with afunctional material. As indicated earlier, a surprising discovery wasthat functional materials can often be attached to azlactone-functionalbeads of the invention in solvents such as water that have heretoforebeen thought of as being reactive with azlactones. "Material" as usedherein means the principal chemical entity that is desired to beattached to a polymer support to accomplish a specific purpose. Statedanother way, "material" means that portion or residue of the "functionalmaterial" which actually performs the complexing, catalytic, or reagentend-use. "Functional" for purposes of this invention means that portionof the "functional material" which contains a group that can react withan azlactone. "Functional" groups useful in the present invention arehydroxy, primary amine, secondary amine, and thiol. These groups react,either in the presence or absence of suitable catalysts, with azlactonesby nucleophilic addition as depicted in equation (2) below. ##STR14##wherein R¹, R², R³, n, X, and G are as previously defined.

Depending on the functional group present in the functional material,catalysts may be required to achieve effective attaching reaction rates.Primary amine functional groups require no catalysts. Acid catalystssuch as trifluoroacetic acid, ethanesulfonic acid, toluenesulfonic acid,and the like are effective with hydroxy and secondary amine functionalgroups. Amine bases such as triethylamine,1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), and1,5-diazabicyclo[4.3.0]non-5-ene (DBN) are effective as well for hydroxyand thiol functional groups. The level of catalyst employed is generallyfrom 1 to 10 parts, preferably 1 to 5 parts.

As is apparent to one skilled in the art, specific reaction conditionssuch as solvent, temperature, level of catalyst, etc. vary tremendouslydepending on the functional material that is to be attached. Because ofthe myriad of functional materials that have been or could be attachedto polymer supports, any listing of functional materials beyond thegeneric HXG of equation (2) and FLOW CHART I would be incomplete andsomewhat unnecessary, as the inventive aspects of the present inventiondo not reside with the functional materials.

Having described the invention in general terms, objects and advantagesof the invention are more specifically illustrated by the followingexamples. The particular materials and amounts thereof recited in theexamples, as well as other conditions and details, should not beconstrued to unduly limit this invention.

EXAMPLE 1

This example teaches the preparation of a gel-type polymer of theinvention.

Preparation of Copoly(N,N-Dimethylacrylamide:2-Vinyl-4,4-Dimethylazlactone:Methylene-bisacrylamide) (46:46:8)

Step 1: Preparation of Copoly(N,N-Dimethylacrylamide (DMA):N-Acryloylmethylalanine Sodium Salt (NaAMA):Methylenebisacrylamide(MBA)) (44.6:50.4:7.8): A two-liter creased, round bottomed flaskequipped with a mechanical stirrer (stirring rate ca. 300 rpm), nitrogeninlet, thermometer, and condenser was charged with heptane (1043 mL) andcarbon tetrachloride (565 mL). This solution was stirred and spargedwith nitrogen for 15 minutes. A separate solution was preparedconsisting of a sodium hydroxide solution (6.6 grams; 0.165 moledissolved in 85 mL of water), N-acryloylmethylalanine (AMA) (25.98grams; 0.165 mole), DMA (23 grams; 0.232 mole), MBA (4 grams; 0.026mole), and ammonium persulfate (1 gram; 0.004 mole) and added to theorganic suspending medium. Sorbitan sesquioleate (Arlacel™ 83, ICIAmericas, Inc., Wilmington, Del.) (2 mL) was added and the mixturestirred and sparged with nitrogen for 15 minutes.N,N,N',N'-tetramethyl-1,2-diaminoethane (2 mL) was added and thereaction temperature rose fairly quickly from 21° C. to 33° C. Themixture was stirred at room temperature for three hours. The mixture wasefficiently filtered using a "D" (>21 microns) sintered glass funnel,and the filter cake washed thoroughly and repeatedly with acetone. Afterdrying at 60° C. and <1 Torr. for 12 hours, the dry solid (52 grams) wassieved and separated into four fractions: beads <38 microns, 12.32grams; beads between 38 and 63 microns, 19.83 grams; beads between 63and 90 microns, 4.56 grams; and beads >90 microns, 13.95 grams.Employing an optical microscope arrangement consisting of a NikonNomarski Differential Interference Contrast Microscope, a Dage Newviconvideo camera, a Sony 3/4" video recorder, and a Perceptive Systems, Inc.digital image processor with accompanying software, it was determinedthat the 38-63 micron sample consisted of quite spherical beads (averageaspect ratio=0.87) which swell in water with an accompanying increase involume of from 35-50%.

Step 2: Cyclization to Copoly(DMA:2-vinyl-4,4-dimethylazlactone(VDM):MBA) (46:46:8): Acetic anhydride (100 mL) was added to 15.1 gramsof the 38-63 micron beads prepared in Step 1. The mixture was heated to100° C. for two hours. After cooling and filtering, the beads wereplaced in a Soxhlet extraction apparatus and were extracted with ethylacetate for 16 hours. After drying at 60° C. and <1 Torr., the beadsweighed 12.6 grams. Infrared analysis (Nujol mull) showed a strongazlactone carbonyl absorption at about 1820 cm⁻¹.

EXAMPLE 2

This example teaches use of the reaction product of 1,2-diaminoethaneand VDM as a crosslinking monomer.

Preparation ofN,N'-bis(2-acrylamido-2-methylpropionyl)-1,2-diaminoethane

A 100 mL, three-necked, round bottomed flask equipped with a magneticstirring bar, a dropping funnel, thermometer, and condenser was chargedwith VDM (13.9 grams; 0.10 mole) and tetrahydrofuran (50 mL). A solutionof 1,2-diaminoethane (3.0 grams; 0.05 mole) in tetrahydrofuran (10 mL)was added dropwise such that the temperature did not exceed 30° C. Afterstirring overnight the reaction mixture was filtered to remove a whitesolid which after washing with hexane and drying at <1 Torr. weighed15.8 grams (93% yield). The solid melted at 207°-210° C. and exhibitedsatisfactory elemental analyses and spectral characteristics for thedesired material, which is the compound of Formula VIII in thespecification.

Preparation ofCopoly(N,N-Dimethylacrylamide:2-Vinyl-4,4-Dimethylazlactone:N,N'-Bis(2-Acrylamido-2-MethylPropionyl)-1,2-Diaminoethane)(55:42.7:2.3)

The two-step procedure of EXAMPLE 1 was utilized except MBA was replacedby the above prepared crosslinking monomer (1.0 gram; 0.003 mole). Asample (15.1 grams) of the intermediate carboxylate-functional polymerwas treated with acetic anhydride to yield, after washing and drying,11.0 grams of the azlactone-functional polymer.

EXAMPLE 3

This example teaches the reaction of a gel-type polymer and a relativelylow molecular weight, intrapolymer support-diffusible functionalmaterial. The example further teaches a procedure for quantitativedetermination of azlactone groups.

The procedure is a variation of a quantitative analysis of isocyanatesand isothiocyanates using n-butylamine (cf. S. Siggia, "QuantitativeOrganic Analysis via Functional Groups", John Wiley & Sons: New York, p.558 (1963)). Generally, the procedure involves treatment of theazlactone-functional beads with standard triethylamine inN,N-dimethylformamide (DMF) to react with and determine theconcentration of any uncyclized carboxyl groups. To another sample ofbeads, excess standard n-butylamine in DMF is added and shaken for 24hours at room temperature. The excess concentration of n-butylamine isthen determined by potentiometric titration with standard acid as anindirect measure of the concentration of azlactone groups. Using thismethod with the beads of EXAMPLE 2, three separate determinations showedminimal, i.e., <0.3 milliequivalents/gram of resin, carboxyl content andan average azlactone content of 2.2 meq/g. Theoretical azlactone contentwas 3.1 meq/g. Therefore, over 70% of the theoretical azlactone groupshad formed and were accessible by the n-butylamine functional material!

EXAMPLE 4

This example further teaches the reaction of a gel-type polymer with arelatively small functional material, N-(3-aminopropyl)morpholine, butin an aqueous reaction solvent. Determination of reactable azlactonecontent is made by measuring the increase in % nitrogen of the reactedbeads. This procedure is more time consuming than the quantitativeanalysis method outlined in EXAMPLE 3, but comparison of the resultsserves as a check on the accuracy of the titration method.

A gel-type polymer consisting of DMA:VDM:MBA (53.8:41.7:4.5) wasprepared as in EXAMPLE 1; the theoretical % nitrogen present in thebeads should be 12.6%; experimentally observed using a Kjeldahl methodwas 12.1%.

The azlactone-functional beads (1.44 grams; containing approximately0.004 mole of azlactone groups), N-(3-aminopropyl)-morpholine (0.80gram; 0.0055 mole), and 15 mL of a standard aqueous pH 9 buffer solutionwere placed in a 100 mL, round bottomed flask and stirred at roomtemperature. After four hours the beads were filtered, washed repeatedlywith deionized water, and dried at 60° C. and <1 Torr. The resultingadduct possessed a nitrogen content of 13.8%. Theoretically, theincrease in nitrogen should have been 17.4%. The experimentally observedincrease of 12.3% again indicates that 70% of the azlactone groups hadformed and reacted. This result is in excellent agreement with thetitration procedure result of EXAMPLE 3. Furthermore, the resultindicates that measurable hydrolysis in the aqueous pH 9 buffer solutiondid not occur and that virtually quantitative attaching reactions cantake place in aqueous media at an elevated pH.

EXAMPLES 5-7

These examples illustrate how polymer bead size can be controlled by thelevel of crosslinking monomer.

The procedure of Step 1 of EXAMPLE 1 was utilized to prepare thecarboxylate-functional beads of the following examples. Average particlediameters were determined using an optical microscope equipped with aZeiss IBAS™ Image Analyzer. It is apparent that as the level ofcrosslinker increases the particle diameter decreases.

    ______________________________________                                                                Wt %    Average particle                              EX-    Monomer wts. (g) cross-  Diameter                                      AMPLE  DMA     NaAMA    MBA   linker                                                                              (micrometers)                             ______________________________________                                        5      24      24       2     4     67.5                                      6      23      23       4     8     42.2                                      7      21      21       8     16    32.4                                      ______________________________________                                    

EXAMPLES 8-10

These examples teach the preparation of macroporous polymers of theinvention. They furthermore teach utilization of a co-solvent tofacilitate dissolution of the crosslinking monomer.

The method of EXAMPLE 1 was utilized except the monomers and initiatorwere dissolved in water (75 grams) and DMF (30 grams). The azlactonecontent was determined utilizing the titration procedure of EXAMPLE 3.

    __________________________________________________________________________                                    Step 2 beads                                         Step 1 changes                                                                             Average particle                                                                          azlactone content                                                                      (meq/g)                              EXAMPLE                                                                              DMA NaAMA                                                                              MBA diameter (micrometers)                                                                    theoretical                                                                            Measured                             __________________________________________________________________________    8      34.02                                                                             4.48 12.5                                                                              26.0        0.5      0.29                                 9      30.55                                                                             8.95 12.5                                                                              20.5        1.0      0.52                                 10     27.08                                                                             13.42                                                                              12.5                                                                              25.0        1.5      1.10                                 __________________________________________________________________________

EXAMPLE 11

This example teaches the preparation of a macroporous polymer withN-methacryloylmethylalanine sodium salt (NaMMA) instead of NaAMA. Theresulting azlactone-functional bead of Formula V was formed with R¹═CH₃.

The procedure of EXAMPLE 9 was utilized except that NaMMA (9.65 grams)was substituted for the NaAMA. The resulting azlactone-functional beadswhich were formed after treatment with acetic anhydride had an averageparticle diameter of 22.4 micrometers and an azlactone functionality of0.68 meq/gram.

EXAMPLE 12

This example teaches the preparation of a macroporous polymer withN-vinylpyrrolidone as the water soluble monomer component. The procedureand monomer charges of EXAMPLE 9 were utilized except the DMA wasreplaced by N-vinylpyrrolidone. The average particle diameter of thebeads resulting from Step 1 was 19.3 micrometers. Cyclization affordedazlactone-functional beads which possessed a strong azlactone carbonylabsorption band at about 1820 cm⁻¹ in the infrared.

EXAMPLE 13

This example teaches the synthesis of a six-membered ring azlactone(2-oxazin-6-one) functional polymer bead. The procedure of EXAMPLE 9 wasutilized except 3-acrylamido-3-methylbutyric acid sodium salt (9.65grams) was utilized instead of NaAMA. After cyclization the2-oxazin-6-one functional beads possessed an average diameter of 28.5micrometers and a functional level of 0.16 meq/g.

EXAMPLE 14

This example teaches the reaction of an azlactone-functional polymerbead with a protein functional material. Protein A (from Staphylococcusaureus) is a commercially available material (Pharmacia Fine Chemicals;Division of Pharmacia, Inc., Piscataway N.J.). The protein, as well asthe protein immobilized on a Sepharose™ support, has manifold uses.Pharmacia Fine Chemicals has issued two publications: "Protein A (S.aureus): Selected Applications of Free and Labeled Protein A", revisededition (January 1982) and "Protein A-Separose CL-4B: SelectedApplications References", revised edition (January 1982); bothpublications are herein incorporated by reference.

Preparation of Radio-labeled Protein A

Protein A (2.5 mg) (Genzyme Corp., Boston, Mass.) was dissolved in 10 mMpotassium phosphate buffer (pH 7.0; 0.6 mL) and two Iodo-beads (aninsoluble form of chloramine T; Pierce Chemical Co., Rockford, Ill.)were added to catalyze the addition of iodine to tyrosine residues. Thereaction was initiated by the addition of 1.5 mCi of NaI (carrier-free¹²⁵ I, New England Nuclear Co., N. Billerica, Mass.). The reaction wasincubated at 20° C. for 30 minutes with vigorous manual shaking at fiveminute intervals. Protein A (both iodinated and unmodified forms) wasseparated from NaI by elution through a Pharmacia PD-10 size exclusioncolumn in the same phosphate buffer. The fractions which containedprotein were combined, aliquotted, and frozen at -15° C. until used.Specific radioactivity on day 0 was 154,000 cpm/ug. All subsequentcalculations were corrected for the radioactive half-life of ¹²⁵ I of 60days. Radioactive Protein A was not used beyond six weeks afteriodination.

Reaction of the Radio-labeled Protein A with an Azlactone-FunctionalBead

The azlactone-functional polymer utilized was that prepared in EXAMPLE5. The polymer beads (0.010 gram) were placed in a centrifuge tube andwere covered with a solution consisting of the labeled Protein Apreparation above (100 ul) and 400 ul of a phosphate buffer solution (pH7.5). The mixture was shaken gently at room temperature for 90 minutes.The tube was centrifuged, and the original supernatant and fivesuccessive washes (1 ml of pH 7.5 buffer) were collected and their ¹²⁵ Icontent determined using a Packard Auto-Gamma Scintillation SpectrometerModel 5230. The original supernatant exhibited 42,415 cpm (abovebackground); first wash: 6722; second wash: 836; third wash: 202; fourthwash: 48; and fifth wash: 18 cpm. Ethanolamine (400 microliters of 0.5 Min pH 7.5 phosphate buffer) was added and shaken with the beads for 90minutes to react with all the remaining azlactone residues. Finally,after an additional four washes with buffer solution the beads and thereaction vessel were counted and exhibited 7002 and 1865 cpm,respectively. This correlates to a level of 2.54 micrograms of ProteinA/10 mg of beads.

A CONTROL was conducted in the same manner except the order of additionof Protein A and ethanolamine was reversed. The CONTROL exhibited alevel of 0.14 microgram of Protein A/10 mg of beads.

In a similar fashion, the effects of the catalyst DBU were examined,with the DBU (25 microliters) being added to the initial Protein Abuffer solution. The result was a level of 3.90 micrograms of ProteinA/10 mg of beads.

EXAMPLE 15

This example illustrates that the Protein A is not just adsorbed oradhering to the beads in some fashion but is actually covalently boundto the polymer beads. The amount of covalently bound protein may beestimated by determining the amount of protein resistant to sodiumdodecylsulfate (SDS) treatment. SDS denatures protein so that only thosemolecules which are covalently bound will remain attached to the beads.

In this experiment, the polymer beads of EXAMPLE 14 (having 3.90micrograms Protein A/10 mg of beads) (0.010 gram) were incubated with 1%SDS (500 microliters) at 37° C. for two hours, followed bycentrifugation, and five buffer (550 microliters; pH 9.5) washes.Analysis of the radioactivity of the beads revealed that 73% of theprotein remained attached to the beads.

EXAMPLE 16

This example illustrates that the Protein A attached to the polymerbeads remains active to accomplish a specific function and is notdenatured in the attaching process.

Biologically active Protein A can be assayed by determining the amountof antibody which it can bind. Antibody (IgG) conjugated with an enzymemarker, alkaline phosphatase was purchased from Cooper Biomedical(Malvern, Pa.).

In this experiment, 1.0 mg of the polymer beads of EXAMPLE 5 werereacted with unlabeled Protein A and ethanolamine as described inEXAMPLE 14. After the washing steps, Protein A and CONTROL beads wereresuspended in the alkaline phosphatase assay solution (0.1 M sodiumglycinate, 1.0 mM ZnCl₂, 1.0 mM CaCl₂, 6.0 mM p-nitrophenyl phosphate,pH 10.4) and rocked continuously to promote mixing. Every 10 min. theabsorbance of the supernatant solution was determined at 405 nm. Theabsorbance increased linearly at 5 to 15 times the CONTROL rate,depending on the amount of immobilized Protein A. This showed that theprotein remained active.

EXAMPLE 17

This example teaches that the attaching reaction with a protein inaqueous media is rapid.

The beads of Example 6 were reacted with radiolabeled Protein A asdescribed in Example 14 except that the quenching and washing steps wereinitiated at various times from 5-180 min. The "zero time" was preparedby addition of the quencher ethanolamine first. The reaction wasperformed in pH 8.5 sodium pyrophosphate buffer with DBU. It wasobserved that at 5 minutes 1.34 micrograms of Protein A/10 mg of beadswere bound. This was 80% by weight of the amount bound at 180 minutes.

Various modifications and alterations of this invention will becomeapparent to those skilled in the art without departing from the scopeand spirit of this invention, and it should be understood that thisinvention is not to be unduly limited to the illustrative embodimentsset forth herein.

We claim:
 1. A carboxylate-functional polymeric bead which is theaqueous phase reaction product of:(i) 0 to 98 parts of free radicallyaddition polymerizable, water soluble monomers, (ii) 1 to 99 parts of awater soluble salt of N-(meth)acryloylamino acid, and (iii) 1 to 30parts of at least one crosslinking monomer.
 2. A carboxylate-functionalbead according to claim 1 having units of the formula: ##STR15## whereinR¹, R², R³ and n are as previously defined, andM is a water-solubilizingcation, and n=0 or
 1. 3. The carboxylate-functional bead according toclaim 2 wherein R¹, R², and R³ are methyl and n=0.
 4. A methodcomprising the steps of:(a) polymerizing together(i) 0 to 98 parts of atleast one free radically addition polymerizable, water soluble monomer,(ii) 1 to 99 parts of at least one water soluble salt ofN-(meth)acryloylamino acid, and (iii) 1 to 30 parts of at least onecrosslinking monomer, (b) isolating the resulting carboxylate-functionalpolymer beads.
 5. The method according to claim 4 further comprising thesteps of:(c) reacting said carboxylate-functional beads with a cyclizingagent, and (d) isolating the resulting azlactone-functional polymerbeads.
 6. The method according to claim 5 further comprising the stepsof:(e) reacting the azlactone-functional polymer beads with a functionalmaterial capable of reacting with said azlactone in a ring-openingreaction, and (f) isolating the resulting adduct beads.
 7. The methodaccording to claim 6 wherein said step (e) takes place in aqueoussolution.
 8. The method according to claim 6 wherein said functionalmaterial is selected from the group consisting of proteins, catalysts,reagents, and dyes.